Transmembrane signaling typically requires a cell surface receptor, a G protein, and an effector enzyme. RGS proteins modulate signaling, by accelerating GTP hydrolysis and G protein inactivation. Signaling can also be modulated through post- translational protein modifications. This proposal will test the hypothesis that RGS and G proteins are also regulated post- translationally. These experiments will be carried out in the yeast S.cerevisiae, which expresses a G protein signaling apparatus similar to those in humans. The use of the yeast system will allow in vitro biochemical methods and in vivo genetic strategies to be used in a coordinated manner, to address the following four aims: Aim 1: How is the RGS protein regulated? Previously we used mass spectrometry to show that the RGS in yeast (Sst2) is phosphorylated at Ser-380 and Ser-539 in vivo. Ser-539 is phosphorylated in response to pheromone stimulation, requires a MAP kinase, and stabilizes the protein. Ser-380 phosphorylation has not been characterized. An expression array library will be used to identify the kinase that phosphorylates Ser-380. Kinase-disruption and phosphorylation-site mutants will then be used to determine how this modification alters G protein catalytic activity in vitro, and G protein signaling activity in vivo. Aim 2: How is the G protein a subunit regulated? It is established that the Galpha in yeast (Gpa1) is ubiquitinated, but the location and functional role of this modification are unknown. Mass spectrometry will be used to identify the ubiquitinated amino acid. Mutants in the ubiquitination and proteolysis pathways will be used to determine the mechanism of G protein degradation. Finally, gene disruption and ubiquitination-site mutants will be used to determine how this process alters G protein activity. Aim 3: How is the G protein B subunit regulated? It is known that the Gbeta in yeast (Ste4) is phosphorylated in response to pheromone stimulation. There is indirect evidence that Ste4-phosphorylation contributes to signal desensitization. Mass spectrometry will be used to identify the site of phosphorylation. The expression array library will be used to identify the Ste4-kinase. Kinase-disruption and phosphorylation-site mutants will then be used to determine how this modification alters G protein activity in vitro and in vivo. Aim 4: What other modifications regulate RGS and G proteins? With recent advances in mass spectrometry, it should now be possible to identify all modifications of a protein of interest. The likelihood that the RGS and G protein undergo additional (as yet unidentified) modifications will be tested. The molecular and cellular consequences of each modification will be evaluated using standard assays of RGS and G protein function.